1. Field of the Invention
This invention relates to methods for the use of compounds to potentiate the effects of gamma-aminobutyric acid (GABA) in mammalian tissues which contain receptors which will bind GABA. In particular, the invention relates to the use of certain non-steroidal agents which enhance the response of GABA.sub.A receptors to GABA and are, therefore, useful as GABAergic agents.
2. History of the Prior Art.
Gamma-aminobutyric acid, commonly known as GABA, is an amino acid which serves as one of the major neurotransmitters in the mammalian central nervous system, particularly the brain.
In general, neurotransmitters are responsible for regulating the conductance of ions across neuronal membranes. At rest, the neuronal membrane possesses a potential or membrane voltage of approximately -80 mv, the interior being negative with respect to the exterior of the cell. The potential is the result of ion balance (e.g., K.sup.+, Na.sup.+, Cl.sup.-, organic anions) across the neuronal membrane, which is semi-permeable. Neurotransmitters are stored in presynaptic vesicles and are released under the influence of neuronal action potentials. When released into the synaptic cleft, an excitatory chemical transmitter such as acetylcholine will cause membrane depolarization (a decrease from its resting value of -80 mv). This effect is mediated by post-synaptic hetero-oligomeric proteins--nicotinic receptors--which are stimulated by acetylcholine to increase membrane permeability to Na.sup.+ and Ca.sup.2+ ions. The reduced membrane potential stimulates neuronal excitability and results in the generation of a post-synaptic action potential, which may cause excitation or inhibition in other nerve cells.
The profound influence of GABA on the central nervous system is related to the presence of GABA receptors in up to 40% of the neurons in the brain alone. GABA regulates the excitability of individual neurons by regulating the conductance of ions across the neuronal membrane. For example, GABA interacts with its recognition site on GABA.sub.A receptors, resulting in an increase in membrane permeability to chloride ions that renders the neuron less susceptible to further stimulation. Thus, GABA receptors have been implicated in the mediation of mental depression, anxiety, seizures and responses to other stresses on the central nervous system.
Flufenamic acid, meclofenamic acid, mefenamic acid, and niflumic acid are all derivatives of N-phenyl-anthranilic acid which are commonly referred to as "fenamates". Fenamates have been used in Europe and Japan as non-steroidal anti-inflammatory agents ("NSAIDs"), but have not been used widely in the United States because of a relatively high incidence of gastrointestinal side effects observed in clinical trials (see, e.g., Wolfe, et al., Arch. Intern. Med., 136:923-925, 1976).
At the molecular level, the fenamates are known to inhibit anion transport through certain ion channels. For example, mefenamic acid has been shown to inhibit current responses to GABA in disassociated rat neuronal cells (Shirasaki, et al., Brain Res., 562:329-331, 1991). Niflumic acid has been shown to be a fairly potent inhibitor of anion transport in erythrocytes (Cousin, et al., J. Membr. Biol. 46:125-153, 1979). In cultured spinal cord neurons, niflumic and flufenamic acids inhibit the responses of N-methyl-D aspartate receptors to their ligand (Lerma, et al., Mol. Pharmacol., 41:217-22, 1992) and also inhibit Ca.sup.2.+-. activated Cl.sup.- channels in the oocytes of the frog Xenopus laevis.
The observations concerning the effect of fenamates on Xenopus laevis oocytes are of interest because Xenopus oocytes are used extensively as an expression system for mammalian neurotransmitter receptors and ion channels, including those which interact with GABA. Generally, the products of the Xenopus oocyte expression system are structurally and functionally like their corresponding native receptor or ion channel in most respects. For example, poly (A).sup.+ RNA isolated from rat cerebral cortex or chick optic lobe expresses GABA.sub.A receptor subunits that assemble to form GABA-activated Cl.sup.- channels. The expressed receptors generally respond to ligands which are known to be potentiatory or inhibitory agents for GABA receptors in the same manner as the native receptors (see, e.g., Parker, et al., J. Neuroscience, 6:2290-2297, 1986; Parker, et al., Proc. R. Soc. Land. B., 233:201-216, 1988; Woodward, et al., Mol. Pharmacol., 41:89-103, 41:1107-115, and 42:165-173 (all 1992); and Sigel, et al., Proc. Nat'l. Acad. Sci., USA, 85:6192-6196, 1988).